Functional monomers for molecular recognition and catalysis

ABSTRACT

The present invention refers to new classes of polymerisable monomers, to molecularly imprinted polymers obtainable by polymerisation of at least one of the monomers and a crosslinking monomer in the presence of a template molecule. The obtained polymers may be used for separation of enantiomers, diastereomers of the template molecule, and also for separation of the template molecule or template molecule analogues from structurally related compounds.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to new polymerisable functionalmonomers, to molecularly imprinted polymers obtainable by polymerisationof at least one of the monomers and a crosslinking monomer in thepresence of a template molecule. The invention also relates to the useof said polymers.

BACKGROUND ART

[0002] In the fields of medical, dietary, environmental and chemicalsciences there is an increasing need for the selective separation ofspecific substances in complex mixtures of related substances. The endgoal can be the preparative isolation of a certain compound or compoundsor measurements of their concentration. Molecularly imprinted polymers(MIPs) often exhibit a high selectivity towards their substrate inanalogy with antibody-antigen complementarity. (1) The technique showspromise in chiral separations of, for example, amino acid derivatives,peptides, phosphonates, aminoalcohols and beta-blocking compounds,affinity chromatography of nucleotides and the DNA-bases, as well assubstitutes for antibodies in immunoassays for commercial drugs. (2)Molecular imprinting (MI) consists of the following key steps (FIG. 1):(1) Functional monomers are allowed to interact reversibly with atemplate molecule in solution. (2) The hereby formed template assembliesare copolymerized with a crosslinking monomer resulting in a crosslinkednetwork polymer. (3) The template is displaced and the materials can beused for selective molecular recognition of the corresponding compound.If these are crushed and sieved they can be packed in a chromatographiccolumn and used for chromatographic separation of the template fromstructurally related analogues. Analytical as well as preparativeapplications are possible. Preparative applications can be separation ofa compound from a complex mixture of structurally related compounds andisolation of the compound. This can be through an affinitychromatographic procedure where pH, ion strength or solvent gradientscan be used in order to control the strength of interaction with thestationary phase. The separation can target enantiomers or diastereomersin a mixture of enantiomers or diastereomers of one or many compounds.Analytical applications can in addition to the above mentionedseparations be: competitive binding assays, chemical sensors orselective sample enrichments. (3)

[0003] Currently the most widely applied technique to generatemolecularly imprinted binding sites is represented by the noncovalentroute. (4) This makes use of noncovalent self-assembly of the templatewith functional monomers prior to polymerisation, free radicalpolymerisation with a crosslinking monomer and then template extractionfollowed by rebinding by noncovalent interactions. Although thepreparation of a MIP by this method is technically simple it relies onthe success of stabilisation of the relatively weak interactions betweenthe template and the functional monomers. Stable monomer-templateassemblies will in turn lead to a larger concentration of high affinitybinding sites in the resulting polymer. The materials can be synthesizedin any standard equipped laboratory in a relatively short time and someof the MIPs exhibit binding affinities and selectivities in the order ofthose exhibited by antibodies towards their antigens. Most MIPs aresynthesized by free radical polymerisation of functional monounsaturated(vinylic, acrylic, methacrylic) monomers and an excess of crosslinkingdi- or tri-unsaturated (vinylic, acrylic, methacrylic) monomersresulting in porous organic network materials. These polymerisationshave the advantage of being relatively robust allowing polymers to beprepared in high yield using different solvents (aqueous or organic) andat different temperatures. This is necessary in view of the varyingsolubilities of the template molecules.

[0004] The most successful noncovalent imprinting systems are based oncommodity acrylic or methacrylic monomers, such as methacrylic acid(MAA), crosslinked with ethyleneglycol dimethacrylate (EDMA). Initially,derivatives of amino acid enantiomers were used as templates for thepreparation of imprinted stationary phases for chiral separations(MICSPs) but this system has proven generally applicable to theimprinting of templates allowing hydrogen bonding or electrostaticinteractions to develop with MAA. (5) The procedure applied to theimprinting with L-phenylalanine anilide (L-PA) is outlined in FIG. 1. Inthe first step, the template (L-PA), the functional monomer (MAA) andthe crosslinking monomer (EDMA) are dissolved in a poorly hydrogenbonding solvent (diluent) of low to medium polarity. The free radicalpolymerisation is then initiated with an azo initiator, commonlyazo-N,N′-bis-isobutyronitrile (AIBN) either by photochemical homolysisbelow room temperature or thermochemically at 60° C. or higher. In thefinal step, the resultant polymer is crushed by mortar and pestle or ina ball mill, extracted in a Soxhlet apparatus and sieved to a particlesize suitable for chromatographic (25-38 μm) or batch (150-250 μm)applications. The polymers are then evaluated as stationary phases inchromatography by comparing the retention time or capacity factor (k′)of the template with that of structurally related analogues.

[0005] A number of compound classes are only poorly recognized bypolymers prepared using the present imprinting protocols. Furthermorethe binding strength between the functional monomer and the template isoften insufficient, leading to a low sample load capacity andsignificant non-specific binding. There is therefore a need for thedevelopment of new functional monomers binding more strongly to thetemplate and allowing recognition of new compound classes. For instancemonomers designed to bind carboxylic-, phosphoric- or phosphonic-acidtemplates are needed. A few examples are given here. One goal in theanalytical applications is the development of imprinted polymers capableof strongly binding nucleotides and discriminating between the fourbases. This may lead to new methods for sensitive detection of modifiedDNA bases and may thus find use in early cancer diagnosis. However, thisrequires development of new functional monomers capable of bindingphosphate thereby increasing the organic solubility of the template.Moreover recognition of adducts of the four DNA bases formed uponexposure of DNA to oxidizing or alkylating agents is one important goalin the development of methods aiming at early diagnosis or prediction ofcancer risks. However, this requires development of new functionalmonomers capable of binding purine and pyrimidine bases with highfidelity. In chiral separations and for analytical applications usingimprinted polymers there is also a need for functional monomers capableof strongly binding compounds with weakly acidic hydrogens such as inalcohols, imides, sulfonamides, phosphonamides, ureas includingimportant classes like carbohydrates, sulfonylureas, hydantoins,barbiturates, purine, pyrimidine and pteridine bases. If these wereavailable, higher selectivities and capacities for the target compoundor enantiomer could be expected. Weakly polar or nonpolar compounds arealso an important group of targets that are difficult to bind with highselectivity. Among these are highly important and relevant analytes suchas dioxines (tetrachlorodioxodibenzene, TCDB), polyaromatic hydrocarbons(PAHs), aldehydes, halogenated hydrocarbons and phosphonates (such asthose found in nerve agents and insecticides).

[0006] This invention describes the synthesis and use of new classes offunctional monomers for molecular imprinting. Two classes are based onthe amidine-functional group and can be synthesized with variousbasicities, hydrophobicities and chiralities. Amidine functionalizedmonomers have been previously used in molecular imprinting in the formof derivatives of 4-vinylbenzamidine. (16) These are synthesized inseveral steps and can only be produced with a limited structuralvariation. Also no chiral amidine monomers have been reported for use inmolecular imprinting. This invention introduces a new class of amidinebased monomers, formamidines, that are accessible in high yield in onlya few synthetic steps. The monomers are suited for imprinting of avariety of functional groups including carboxylic and phosphorous acids,alcohols, imides, sulfonamides, phosphonamides and ureas which can beachiral or chiral. Furthermore the synthetic route allows theirproperties such as pKa values and polarities to be easily tuned. Theother class of amidine based monomers are chiral amidines thatpotentially will enhance the recognition of chiral substrates. Thisbasic benzamidine unit has previously been used as chiral shift reagentsin determination of optical purity by NMR. (17) Another class offunctional monomers are vinyl-methacrylolyl- or acryoloyl-based alkyl oraryl diamines (4) which was previously used as a chiral shift reagentfor diols. This monomer is designed to bind carbohydrates.

[0007] Other classes of polymerisable functional monomers of the presentinvention are receptor analogue monomers, urea based monomers, thioureabased monomers, purine or pteridine based monomers, acrylamido ormethacrylamido pyridine monomers, aminovinylpyridine monomers, stronghydrogen bond accepting monomers that are unable to function as hydrogenbond donors, such as hexamethyl phosponamide based monomers, and stronghydrogen bond donating monomers that are unable to function as hydrogenbond acceptors, such as N,N′-disubstituted phenyl urea monomers, asdefined below.

[0008] This invention describes the synthesis and use of new classes offunctional monomers for molecular imprinting. They will be describedbelow together with a number of non-restricting examples of theirsynthesis and use.

[0009] The invention will now be described in more detail giving anumber of non-restricting examples:

SUMMARY OF THE INVENTION

[0010] Thus, the present invention relates to a polymerisable functionalmonomer, wherein said monomer is selected from at least one of thefollowing:

[0011] a) a formamidine monomer;

[0012] b) a chiral amidine monomer;.

[0013] c) a vinyl-methacrylolyl or acryoloylbased alkyl or aryl diamine;

[0014] d) a receptor analogue monomer of one of the following formulas

[0015] wherein each R¹ is independently selected from the groupconsisting of H and CH₃ and R² is independently selected from the groupconsisting of alkyl and aryl;

[0016] e) an urea monomer of the formula

[0017] wherein each R¹ is independently selected from the groupconsisting of H and CH₃;

[0018] f) a thiourea monomer of the formula

[0019] wherein each R¹ is independently selected from the groupconsisting of H and CH₃;

[0020] g) a purine or pteridine based monomer of one of the followingformulas

[0021] wherein each R is independently selected from the groupconsisting of H and NH₂ and R¹ is selected from the group consisting ofH and CH₃;

[0022] h) an acrylamido or methacrylamido pyridine monomer of one of thefollowing formulas

[0023] wherein each R is independently selected from the groupconsisting of H and CH₃;

[0024] i) an aminovinylpyridine monomer of one of the following formulas

[0025] j) a strong hydrogen bond accepting monomer that is unable tofunction as a hydrogen bond donor, preferably a hexamethyl phosphonamidebased monomer of the formula

[0026] wherein each R is independently selected from the groupconsisting of styryl and vinylbenzyl;

[0027] k) a strong hydrogen bond donating monomer that is unable tofunction as a hydrogen bond acceptor, preferably a N,N′-disubstitutedphenyl urea monomer of the formula

[0028] wherein X, Y and Z are independently selected from the groupconsisting of H, NO₂, CF₃ and halide.

[0029] The monomers a), j) and k) are particularly important aspects ofthe present invention.

[0030] The invention further relates to a polymer obtainable bypolymerisation of at least one of the above monomers a)-k) and acrosslinking monomer in the presence of a template molecule.

[0031] Still further the invention relates to the use of a polymeraccording to the above for separation of enantiomers, diastereomers ofthe template molecule, and also for separation of the template moleculeor template molecule analogues from structurally related compounds.

[0032] Yet further the invention relates to the use of said polymer forcatalysing chemical reactions such as esterolysis, amidolysis, estersynthesis or amide synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 illustrates molecular imprinting with L-phenylalanineanilide (L-PA).

[0034]FIG. 2 illustrates several monomers according to the invention.

PREFERRED EMBODIMENTS OF THE INVENTION

[0035] The invention will now be described in more detail with referenceto a group of non-limiting examples.

[0036] According to one aspect of the present invention the amidinemonomer is a formamidine.

[0037] In one aspect of the invention the amidine monomer is a cyclicamidine.

[0038] According to one aspect the cyclic amidine is formed from oneenantiomer of cyclohexanediamine or substituted cyclohexanediamine.

[0039] According to another aspect the cyclic amidine is formed fromethylenediamine, 1,2-diphenyl-1,2-ethylenediamine or a 1,3-diamine.

[0040] The template molecule used in the polymerisation according to thepresent invention can be selected from the group consisting of anucleotide or oligonucleotide, a polyelectrolyte such as a polysulfonicor polycarboxylic or mixed polysulfonic/polycarboxylic acid, acarboxylic acid, a phosphonic acid, a phosphoric or phosphinic acid, asulfuric, sulfonic, sulfinic acid, an alcohol, an imide, a thiol, aketone, an amide, a sulfonamide, a phosphonamide, a hydantoin, abarbiturate, an ether, a polyaromatic hydrocarbon, a phosphonate, analdehyde.

EXAMPLES

[0041] 1. Benzamidines and Chiral Amidines

Example 1

[0042] The chiral amidine monomer (1) is synthesized starting with a4-bromobenzonitrile or another bromoarylnitrile, or a substitutedbromoarylnitrile which is reacted to form the ethylimidate through aPinner synthesis followed by reaction with a diamine that can beethylenediamine, 1,2-diphenyl-1,2-ethylendiamine, (+) or (−)cyclohexyldiamine, a substituted chiral cyclohexyldiamine or a1,3-diamine. After protection of the basic nitrogen a vinyl group isintroduced by Pd-catalysed reaction between the protected amidine andvinyltributyltin (Stille coupling). After deprotection the amidinemonomer is obtained.

Example 2

[0043] The amidine monomer is synthesized starting with4-acetobenzonitrile or substituted 4-acetobenzonitrile which is reactedto form the ethylimidate through a Pinner synthesis followed by reactionwith a diamine that can be ethylenediamine,1,2-diphenyl-1,2-ethylendiamine, (+) or (−) cyclohexyldiamine, asubstituted chiral cyclohexyldiamine or a 1,3-diamine. After protectionof the basic nitrogen a Wittig reaction is performed by reacting theamidine with the corresponding methylylid. The final product is thenobtained by deprotection of the amidine.

Example 3

[0044] Here we describe the synthesis of a range of amidine monomers,starting from 4-cyanostyrene, prepared using the classical Pinnersynthesis. The preparation of the chiral amidine monomer (1) isdescribed as an example of the general method.

[0045] Anhydrous ether is saturated with anhydrous HCl over the courseof 1 hour. To this solution are added 4-cyanostyrene and anhydrousethanol. Dry HCl is then bubbled through the solution for a further 3hours, during which time a precipitate begins to form. The solution isleft to stand for 18 hours. The large amount of precipitate forms isfiltered off, washed with anhydrous ether and dried in vacuo over P₂O₅,yielding the intermediate imidate salt in 80% yield. The intermediateimidate salt (1 equ.) is dissolved in anhydrous ethanol, with stirring,under an inert atmosphere. To this solution is addedD-1,2-diaminocyclohexane (1 equ.). The resulting solution is stirred for1 hour at 40° C. The solution is then heated to reflux for a further 2hours and then allowed to cool. The cooled solution is poured ontoaqueous 5% sodium hydrogen carbonate and the solution is then extractedwith dichloromethane. The organic phases are combined, dried overmagnesium sulphate and filtered. Removal of the solvent in vacuo yieldsthe product, monomer (1), as a yellow solid in 70% yield.

[0046] A vast series of vinylbenzamidines may be prepared following theabove procedure by substituting D-1,2-diaminocyclohexane for any desiredamine or diamine.

[0047] 2. Formamidines

[0048] N,N′-disubstituted formamidines, of the general formula shownbelow, are capable of both hydrogen bond donation and acceptance. Thesubstituents R and R¹ may be varied so as to yield monomers with a widerange of basicity. A polymerisable moiety is carried either by R (2a) orby both substituents (cross-linking monomers 2b). Choosing R and R1 fromaromatic based moieties, monomers with relatively low basicity areexpected. However choosing them as aliphatic, for instancevinylbenzylamine, more basic monomers are expected with pKa valuesamounting to those typical for primary amidines (pKa>10). Choosing mixedaliphatic (R) and aromatic (R1), or R and R1 being aromatic withelectron-donating/accepting substitutions, the basicity is expected tobe easily tuned to match the particular application.

[0049] Two examples are given below, one for the preparation of monomersof type 2a and the second for monomers of type 2b.

[0050] The typical procedure for the preparation of monomers of type 2ais described below, using the synthesis of N-styryl-N′-phenylformamidine as an example.

Example 4

[0051] The synthesis is achieved in three steps. The first step is thepreparation of N,N′-diphenyl formamidine (by the reaction of anilinewith triethyl orthoformate). This intermediate is then further reactedwith triethyl orthoformate to produce ethyl N-phenylimidoformate. Thefinal step is the reaction of ethyl phenylimidoformate with4-aminostyrene to give the product.

[0052] Thus, a mixture of triethyl orthoformate (1 equ.) and aniline (2equ.) is heated at reflux for 2 hours. The ethanol evolved during thereaction is then distilled through a fractionating column. The reactionmixture is then allowed to cool, whereupon it solidifies.

[0053] Recrystallisation of the solid from toluene yields N, N′-diphenylformamidine in 80% yield.

[0054] N, N′-diphenyl formamidine (1 equ.) and triethyl orthoformate (2equ.) are mixed and a catalytic amount of dry aniline hydrochloride isthen added. The mixture is heated and ethanol is seen to reflux within afew minutes. The mixture is heated at reflux for 1 hour and then theethanol is distilled. Following the distillation, anhydrous potassiumcarbonate is added. The mixture is shaken and then allowed to stand for2 hours. The reaction mixture is then distilled under reduced pressure.At 93 mmHg, excess triethyl orthoformate is collected at 83-85° C., Thepressure is then reduced to 40 mmHg and ethyl N-phenylimdioformate iscollected at 117° C. in 95% yield.

[0055] A mixture of ethyl N-phenylimdioformate (1 equ.) and freshlydistilled 4-aminostyrene (1 equ.) are slowly heated, whereupon anexothermic reaction sets in. Care should be taken to control thetemperature to below 60° C. After heating at 60° C. for 5 minutes, thereaction is cooled and left at −18° C. for 18 hours. The crude productis isolated and then recrystallised from ethanol to yield the product,N-styryl-N′-phenyl formamidine (2a) in 55% yield.

Example 5

[0056] The typical procedure for the preparation of monomers of type 2bis described below, using the synthesis of N,N′-distyryl formamidine asan example.

[0057] Thus, a mixture of triethyl orthoformate (1 equ.) and glacialacetic acid (1 equ.) is heated at reflux for 30 minutes. Freshlydistilled 4-aminostyrene (2 equ.) is added dropwise to the refluxingmixture. The reaction mixture is heated at reflux for 45 minutes. Theapparatus is then set for distillation and the reaction mixture heatedsuch that volatile material is distilled. After distillation of volatilematerial has ceased, the reaction mixture is allowed to cool to roomtemperature and is then evacuated for 18 hours (<1 mbar). The crudesolid product is shaken with aqueous sodium carbonate and extracted withdiethyl ether. The combined ethereal extracts are dried over magnesiumsulphate and then evaporated to dryness. Recrystallisation frompetroleum ether 60-80 affords the product (2b) as white crystals.

[0058] 3. Further Amidines

[0059] A wide range of monomeric amidines may also be prepared by themethod of Fuks. (18) This procedure has previously been used to prepareacrylamidines. However, these compounds polymerise not only via additionpolymerisation of the vinylic group, but also via a polyadditionreaction between the amidine and the vinylic group. Thus, acrylamidinesare of little use in the preparation of MIPs. However, the method isgeneral for the preparation of amidines and polymerisable molecules canindeed be obtained.

[0060] Here, we describe the preparation of monomers (3), where any orall of the groups R, R¹ and R² may carry a polymerisable function. Thetypical procedure is described below, using the synthesis ofN-isopropyl-N′-vinylbenzyl-acetamidine (R=^(i)Pr, R¹=vinylbenyl andR²=CH₃) as an example.

Example 6

[0061] Thus, a suspension containing iron (III) chloride (1 equ.) in alarge excess of isopropyl chloride is mixed with acetonitrile (1 equ.)at 0° C. The suspension is stirred for 3 hours. The excess isopropylchloride is removed in vacua to leave a solid residue. This residue istaken up in dichloromethane and a solution of vinylbenzyl amine (0.95equ.) in dichloromethane is added dropwise to the suspension. Themixture is stirred for two hours at room temperature, after which timethe solvent is removed in vacuo and the residue is taken up in water.The mixture is cooled to 0° C. and 30% aqueous sodium hydroxide (4.5equ.) is added. This mixture is extracted with four times with diethylether. The ethereal extracts are combined, dried over magnesiumsulphate, filtered and then reduced in vacuo. The solid isrecrystallised from petroleum ether 40-60 to yield the product (3) in60% yield.

Example 7

[0062] The polymer is synthesized by free radical polymerisation of amixture of any of the amidine monomers prepared as described in Examples1-6, and a crosslinking monomer, that can be for exampleethyleneglycoldimethacrylate, divinylbenzene ortrimethylolpropanetrimethacrylate, in presence of a solvent and atemplate and an initiator, that can be azobisisobutyronitrile. A thirdmonomer, that can be methacrylic acid or fluoromethylacrylic acid canalso be added. The template can be a nucleotide or oligonucleotide, apolyelectrolyte such as a polysulfonic or polycarboxylic or mixedpolysulfonic/polycarboxylic acid, a peptide, a protein, a carboxylicacid, a phosphonic acid, a phosphoric or phosphinic acid, a sulfuric,sulfonic, sulfinic acid, or compounds with weakly acidic hydrogens suchas alcohols, imides, sulfonamides, phosphonamides, ureas includingimportant classes like carbohydrates, sulfonylureas, hydantoins,barbiturates, purine, pyrimidine and pteridine bases. Furthermoreketones may be suitable templates. The template can also be a transitionstate analogue for a chemical reaction. In the case where the templatehas one or more chiral centres the template may be one enantiomer ordiastereomer of the template isomers. After polymerisation the polymeris freed from the template by a washing procedure.

Example 8

[0063] The polymer prepared according to Example 7 can be used forseparation of enantiomers, diastereomers of the template or forseparation of the template or template analogues from structurallyrelated compounds. This can be done by chromatography, capillaryelectrophoresis, capillary electrochromatography, batch modes ormembrane modes. The polymer can further be used for catalysing chemicalreactions such as esterolysis, amidolysis, ester synthesis or amidesynthesis.

[0064] 4. Receptor Analogue Monomers

[0065] Molecules such as (I) have been identified as recognitionelements for a variety of substrates, including barbiturates anddicarboxylic acids. (8) In molecular imprinting an important advantageis gained in using polymerisable versions of these kind of receptors.Many important biomolecules, i.e. peptides, proteins, cofactors andvitamins, contain discrete diacids as structural features that may be inthe form of the amino acids aspartic and glutamic acid. With monomersavailable for selective complexation of these substructures othermonomers can be added in the imprinting step to target other parts ofthe target molecules. One example of this principle is seen for therecognition of the dihydrofolate-reductase inhibitor methotrexate (A).Here we describe the synthesis of analogous molecules containingpolymerisable functions (II a/b/c). The synthesis proceeds through theintermediate (IV), which is central to all the monomers described.

[0066] A further set of monomers (IIIa/b/c) may be prepared by using aterephthaloyl-spacer unit in place of the isophthaloyl-spacer shownabove. These monomers are prepared in the same manner as described belowfor IIa/b/c (the structure of a type IIIc monomer is shown below).

Example 9

[0067] Intermediate (IV)

[0068] To a solution of 2,6-diaminopyridine (10 equ.) and triethylamine(2 equ.) in THF is added dropwise a THF solution of isophthaloyldichloride (1 equ.) at room temperature under an inert atmosphere. Thereaction mixture is stirred for 3 hours, after which time the solvent isremoved in vacuo. The residue is stirred with water and the resultingprecipitate filtered and washed well with more water (to remove excessdiamine and triethylamine hydrochloride). The product is initiallypurified via alumina chromatography (DCM/THF as eluant) and finally byrecrystallisation from THF/heptane. This yields the product(intermediate 10) as a faintly yellow crystalline solid in 79% yield.

[0069] The monomers listed are all products from the reaction ofcompound (IV) with either one equivalent of acid chloride(mono-functional monomers IIa), the further reaction of type IIamonomers with a different acid chloride (mono-functional monomers IIb)or two equivalents of the desired acid chloride (cross-linking monomersIIc). The methods are described below.

Example 10

[0070] Monofunctional Monomers IIa

[0071] Compound (IV) (1 equ.) and triethylamine (1.5 equ.) are dissolvedin THF. To this solution is added dropwise a THF solution of acryloyl-or methacryloyl-chloride (1.5 equ) at room temperature under an inertatmosphere. The reaction mixture is stirred for 2 hours, after whichtime the precipitate is removed by filtration and the filtrate reducedto dryness in vacuo. The residue is washed with 0.05M aqueous NaOH,followed by water. The product is dispersed in 0.05M HCl and anyinsoluble material is removed by filtration. The solution is neutralisedwith NaHCO₃ and then extracted with chloroform. The chloroform extractsare dried (magnesium sulphate), filtered and reduced to dryness invacuo. The solid residue is subjected to silica chromatography and thedesired product is obtained in 30% yield.

Example 11

[0072] Monofunctional Monomers IIb

[0073] Compounds IIb are prepared via the reaction of compounds IIa withthe desired acid chloride, in essentially the same manner described forthe preparation of compounds IIa. The reaction mixture is stirred for 4hours, after which time the precipitate is removed by filtration and thefiltrate reduced to dryness in vacuo. The residue is washed successivelywith aqueous 0.05M NaOH, aqueous 0.05M HCl and water. Recrystallisationfrom THF/hexane gives the products in 20% yield.

Example 12

[0074] Crosslinking Monomers IIc

[0075] Compounds (IV) (1 equ.) and triethylamine (6 equ.) are dissolvedin THF. To this solution is added dropwise a THF solution of acryloyl-or methacryloyl-chloride (4 equ.) at room temperature under an inertatmosphere. The reaction mixture is stirred for 4 hours, after whichtime the precipitate is removed by filtration and the filtrate reducedto dryness in vacuo. The residue is washed successively with aqueous0.05M NaOH, aqueous 0.05M HCl and water. Recrystallisation fromTHF/hexane gives the products in 70% yield.

[0076] Molecules such as (V) have been identified as recognitionelements for dicarboxylate salts, e.g. glutarates. The incorporation ofsuch molecules into molecularly imprinted polymers could prove apowerful tool in the selective uptake of many biological substrates andpharmaceuticals, e.g. the anti-cancer drug methotrexate (which containsa glutamic acid residue). Here we describe the synthesis of analogousmolecules containing polymerisable functions (ureas VIa/b/c andthioureas VIIIa/b/c).

[0077] These syntheses involve the preparation of the functionalisocyanates (VIIa/b/c). Isocyanates VIIa/b may be prepared by the methodof Lieser. (9) This involves the reaction of the appropriate acidchloride with sodium cyanate in ethereal solution. Isocyanate VIIc maybe prepared by the method of Iwakura et al. (10) This involves thereaction of 4-vinylbenzoyl chloride with sodium azide, followed by aCurtius rearrangement of the formed 4-vinylbenzazide to yield isocyanateVIIc.

Example 13

[0078] Monomers VIa/b/c

[0079] These molecules are prepared via the reaction of isocyanatesVIIa/b/c, respectively, with 1,4-xylylene diamine. Thus, a dilutesolution of 1,4-xylylene diamine (1 equ.) in diethyl ether is addeddropwise to a cooled ethereal solution of the desired isocyanate (1equ.). The precipitate is filtered, washed well with diethyl ether andrecrystallised from ethanol to give the desired product in 70% yield.

[0080] Extensions to the above schemes involve the reaction ofisocyanates VIIa/b/c with any desired amine (or diamine), thus forming avast library of polymerisable ureas for use in molecularly imprintedpolymers. There also exist other methods by which polymerisable ureasmay be prepared, in which the preparation of isocyanates VIIa/b/c isprecluded.

[0081] One example is the method of Johnson et al. (1392-1393 (1959)),which proceeds via the dehydrohalogenation of N-propionyl-N′-aryl ureas.These ureas are formed via the reaction of propionyl isocyanate with theappropriate aryl amine. The isocyanate, prepared from allyl amine andN-bromosuccinimide, need not be isolated and the ureas may be formed inthe same flask as the preparation of the isocyanate was effected.

[0082] A further example is the reaction of activated esters, e.g.alk-1-en-2-yl esters, with the sodium salts of N-phenyl ureas in THF.(11) Here, there is no requirement for the preparation of isocyanates atall.

[0083] Syntheses of thioureas involve the preparation of the functionalisocyanates (IXa/b/c), which may be prepared by the methods described inthe following articles:—

[0084] (i) IXa/b—U.S. Pat. No. 2,327,985 (1940) (to Du Pont de Nemours &Co.)

[0085] (ii) IXc—Overberger & Friedman, J. Org. Chem., 30, 1926-1929(1965)

Example 14

[0086] Monomers VIIIa/b/c

[0087] Thiourea monomers VIIIa/b/c, the sulphur analogues of the ureasdescribed above, may be prepared in a similar manner from the desiredisothiocyanate and 1,4-xylylene diamine.

Example 15

[0088] Further Thioureide Monomers

[0089] Again in analogy to the compounds described above, extensions tothe above schemes involve the reaction of isothiocyanates IXa/b/c withany desired amine (or diamine), thus forming a vast library ofpolymerisable thioureas for use in molecularly imprinted polymers.

[0090] 5. Purine and Pteridine Based Monomers

[0091] The hydrogen-bonding capabilities of purinic molecules are wellestablished, e.g. their role in the structure of DNA. Adenines havepreviously been used as templates in the fabrication of MIPs usingacidic monomers as recognition elements. (6) As a “flip-side” to this,polymerisable purinic molecules, will provide recognition elements forthe molecular imprinting of acidic compounds. Furthermore strongcomplexes between adenine and a large number of important targetmolecules have been observed. Examples of these are complexes betweenadenine or derivatives thereof and riboflavin or analogues of riboflavin(B) and with imidic functions such as those found in hydantoins (C) orbarbiturates. With pteridine-based functional monomers additionalhydrogen bond donor/acceptor arrangements are available These type ofmonomers, as well as the purine based monomers, offer multiple sites(faces) for interaction with a given target. The target will thus selectthe interaction site providing the maximum stability of the complex.Thus one or a few functional monomers can be used to target a vast arrayof molecular targets.

Example 16

[0092] 9-vinylbenzyl-aminopurines (Xa/b)

[0093] 6-amino-9-vinylbenzylpurine (9-vinylbenzyladenine) (Xa) and2,6-diamino-9-vinylbenzylpurine (Xb) are synthesised as shown below viathe alkylation of the parent purine with vinylbenzyl chloride (mixtureof 3- and 4- isomers).

[0094] A mixture of adenine (2.70 g, 20 mmol), 9-vinylbenzyl chloride (amixture of 3- and 4-isomers) (3.36 g, 22 mmol) and potassium carbonate(5.52 g, 40 mmol) in dimethylformamide (150 cm³) is stirred at roomtemperature under nitrogen for seven days. The resultant mixture isfiltered and the filtrate was evaporated to dryness in vacuo(temperature below 40° C.) to give a yellow solid which is washed wellwith water.

[0095] The solid is dissolved in chloroform and the solution washed with1 M aqueous hydrochloric acid. The combined aqueous phases are extractedwith chloroform and then basified by the slow addition of 5M aqueoussodium hydroxide with cooling (ice-water bath). The aqueous phase isthen extracted with chloroform. The combined chloroform extracts aredried over magnesium sulphate, filtered and evaporated to give a whitesolid. The solid is purified by column chromatography (9:1chloroform:methanol as eluant) to yield spectroscopically pure6-amino-9-vinylbenzylpurine (Xa) in 30% yield.

Example 17

[0096] 9-vinyl-aminopurines (XIa/b)

[0097] 6-amino-9-vinylpurine (9-vinyladenine) (XIa) and2,6-diamino-9-vinylpurine (XIb) are synthesised as shown below. Thefirst step involves the alkylation of the parent purine with(2-bromoethyl) methyl ether; this is followed by the elimination ofmethanol by the action of potassium t-butoxide.

Example 18

[0098] 9-acryloyl- and 9-methacryloyl-aminopurines (XII)

[0099] These acryloyl and methacryloyl aminopurine derivatives aresynthesised via the reaction of the appropriate aminopurine withacryloyl chloride and methacryloyl chloride respectively, as shownbelow.

Example 19

[0100] Vinylpteridine Monomer (XIII)

[0101] An interesting extension of the above systems is the preparationand use of a pteridine-based monomer. Such an example is2,4-diamino-6-vinylpteridine (XIII). This molecule is synthesised infive synthetic steps, from -chloropyruvaldoxime, in 15% yield followingthe method of Taylor & Kobayashi (J. Org. Chem., 38, 2187-2821 (1973)).

[0102] 6. Aminopyridine Based Monomers

[0103] Pyridine moieties have been used in the fabrication of MIPs withthe aim of providing sites for the recognition of acidic moieties intemplate molecules. Most commonly used have been the commerciallyavailable 2- and 4-vinylpyridines respectively. Acrylamido-(12) andmethacrylamido-(13) pyridines have previously been synthesised and thelatter have been incorporated into MIPs. Here, we describe a new classof aminopyridine monomers with tunable basicity and polarity. These havebeen successfully used in the synthesis of imprinted polymers showinghigh selectivity for molecules containing acidic functional groups.Methyl groups in the ring are used to tune polarity and basicity.

[0104] Acrylamido- and Methacrylamido-Pyridines (XIV-XVIII)

[0105] The monomers XIV to XVIII are all synthesised in essentially thesame manner via the reaction of the appropriate 2-aminopyridinederivative with the desired acid chloride in dichloromethane, withtriethylamine as base. The products are obtained in 50-70% yieldfollowing purification by silica gel chromatography. The preparation ofmonomer XVb is described as an example of the general procedure.

Example 20

[0106] A solution of 2-amino-6-methyl pyridine (1 equ.) andtriethylamine (1.1 equ.) in dichloromethane is cooled to 0° C.Methacryloyl chloride (1 equ.) is added dropwise to the cooled solutionand the solution is allowed to stir for 1 hour, the temperature beingallowed to rise to room temperature. Water is then added and theresultant solution stirred for a further 30 minutes. After this time,the organic layer is separated from the aqueous phase, which is washedthree times with dichloromethane. The combined organic phases are driedover magnesium sulphate, filtered and reduced to dryness in vacuo. Thesolid residue is then purified by silica gel chromatography to give theproduct, XVb, in 60% yield.

[0107] Aminovinylpyridines (XIX-XXI)

[0108] The monomers XIX to XXI are prepared from the correspondingmethyl substituted aminopyridines in two steps. The first is thealkylation of the methyl group via its reaction with n-butyl lithium and(chloromethyl)methyl ether. The resulting compound is then treated withpotassium t-butoxide in the second step to give the vinyl-functionalisedaminopyridine via the removal of methanol. The preparation of monomerXIX is described as an example of the general procedure.

Example 21

[0109] Synthesis of Monomer XIX

[0110] A solution of n-butyl lithium (1 equ.) in hexane andtetrahydrofuran is cooled to 0° C., under an inert atmosphere. To thecooled solution is added dropwise a solution of 2-amino-4-methylpyridine in tetrahydrofuran. The temperature is maintained at 0° C. andthe solution stirred for a further 30 minutes once the addition iscomplete. A solution of chloromethyl)methyl ether (1.1 equ.) intetrahydrofuran is then added dropwise to the reaction solution, withthe temperature maintained at 0° C. After the addition is complete, thesolution is stirred for a further 30 minutes, after which time thereaction is quenched by the addition of water. The reaction mixture isthen partitioned between saturated aqueous sodium hydrogen carbonate anddiethyl ether. The ethereal layer is separated and the aqueous phase iswashed three times with diethyl ether. The ethereal extracts arecombined, dried over magnesium sulphate, filtered and reduced to drynessin vacuo. The residue is then purified by silica gel chromatography toyield the intermediate ether in 70% yield.

[0111] The intermediate ether (1 equ.) is dissolved in tetrahydrofuranand the solution is cooled to −78° C. under an inert atmosphere. To thissolution is added a solution of potassium t-butoxide (2 equ.) intetrahydrofuran. The reaction is stirred for 90 minutes at −78° C.,before the temperature is allowed to rise to room temperature, whence itis stirred for a further 30 minutes. The reaction is then quenched bythe addition of water. The reaction mixture is then extracted threetimes with diethyl ether. The ethereal extracts are combined, dried overmagnesium sulphate and reduced to dryness in vacuo. The residue ispurified by silica gel chromatography to yield the product XIX in 40%overall yield.

[0112] 7. Strong Hydrogen Bond Acceptors

[0113] An example of molecules which can perform only as strong hydrogenbond acceptors is hexamethyl phosphonamide (HMPA). (14) Here we describethe synthesis of polymerisable analogues of HMPA, bearing either one

[0114] polymerisable function (monomers XXII) or three polymerisablefunctions (cross-linking monomer XXIII). This new class of functionalmonomers is of great importance in molecular imprinting for the strongsolubiiization of poorly soluble templates in organic solvents. Itsgeneral use as a polymerisable solubilizing agent will allow theextension of molecular imprinting to other new compound classes.

Example 22

[0115] Functional Monomer XXI

[0116] This monomer is obtained via the reaction of the sodium salt ofpentamethylphosphonamide with vinylbenzylchloride.

[0117] Thus, the sodium salt of pentamethylphosphonamide (1 equ.) andvinylbenzylchloride (1 equ.) are reacted together in toluene at 0° C.The pure monomer XXII is obtained in 60% yield following distillationunder reduced pressure.

Example 23

[0118] Functional Monomers XXIIIa/b

[0119] These monomers are synthesised in two steps. The preparation ofthese tri-functional monomers is explained below, using the preparationof N,N′,N″-tri(vinylbenzyl)-N,N′,N″-tri(methyl) phosphonamide (XXIIIb),as the example.

[0120] To a stirred, cooled, chloroform solution of phosphoryl chloride(1 equ.) is added dropwise pyridine (3 equ.). The temperature of thesolution is maintained at 0° C. during the course of the addition. Thesolution is transferred to a dropping funnel and added dropwise to astirred, cooled (0° C.) chloroform solution of vinylbenzylamine (6equ.). Once the addition is complete, the reaction mixture is refluxedfor 2 hours to ensure complete reaction and then allowed to cool. Anyinsoluble material that appears on cooling is filtered and washed withchloroform. The combined chloroform portions are then reduced to drynessin vacuo. The residue is washed with dilute aqueous HCl and then withwater. The solid was recrystallised from ethanol to yieldN,N′,N″-tri(vinylbenzyl) phosphonamide in 60% yield.N,N′,N″-tri(vinylbenzyl) phosphonamide is converted to the monomerXXIIIb by reaction with methyl iodide and the pure product is obtainedin 50% overall yield, after distillation under reduced pressure.

[0121] 8. Strong Hydrogen Bond Donors

[0122] N,N′-disubstituted phenyl ureas, where (at least) one of thephenyl substituents bears an electron withdrawing group in themeta-position, are examples of compounds capable of hydrogen bonddonation only. (15) These have proved to be exceptionally goodcomplexing agents for hydrogen bond acceptors such as ethers, ketonesand phosphonates. Monomeric versions of analogous structures to be usedin molecular imprinting offers a new way into materials with goodrecognition properties for highly interesting targets such as dioxines(D), nerve agents, PAHs, ketones, aldehydes etc. Polymerisable examplesof such compounds are illustrated below.

Example 24

[0123] Synthesis of Monomers XXIV

[0124] These compounds may be prepared by the reaction of4-aminostyrenes and the appropriate m-substituted phenyl isocyanate. Thetypical procedure is described below, using the synthesis ofN-styryl-N′-(m-nitrophenyl) urea. Thus, 3-nitrophenyl isocyanate (1equ.) and triethylamine (1 equ.) are dissolved in dimethylformamide. Tothe stirred solution is added 4-aminostyrene (1 equ.). The resultantsolution is stirred overnight at room temperature. The red solution isthen poured onto a mixture of crushed ice/water, causing the product toprecipitate. The yellow precipitate is filtered off and washed well withwater. The washed residue is purified by silica gel chromatography togive the product in 70% yield.

[0125] Such monomeric aromatic ureas may also be prepared by thereaction of vinylphenyl isocyanate (described previously—isocyanateVIIb) with substituted aromatic amines. The procedure is essentially thesame as that described in the above paragraph.

Example 25

[0126] The polymer is synthesized by free radical polymerisation of amixture of any of the monomers prepared as described in Example 1-16,and a crosslinking monomer, that can be ethyleneglycoldimethacrylate,divinylbenzene or trimethylolpropanetrimethacrylate, in presence of asolvent and a template and an initiator, that can beazobisisobutyronitrile. A third monomer, that can be methacrylic acid orfluoromethylacrylic acid can also be added. The template can be anucleotide or oligonucleotide, a polyelectrolyte such as a polysulfonicor polycarboxylic or mixed polysulfonic/polycarboxylic acid, a peptide,a protein, a carboxylic acid, a phosphonic acid, a phosphoric orphosphinic acid, a sulfuric, sulfonic, sulfinic acid, or compounds withweakly acidic hydrogens such as alcohols, imides, sulfonamides,phosphonamides, ureas including important classes like carbohydrates,sulfonylureas, hydantoins, barbiturates, purine, pyrimidine andpteridine bases. Furthermore may ketones and aldehydes be suitabletemplates. The template can also be a transition state analogue for achemical reaction. In the case where the template has one or more chiralcentres the template may be one enantiomer or diastereomer of thetemplate isomers. The template can further be a hydantoin (e.g.diphenylhydantoin), flavin (e.g. riboflavin) a dihydropyridine (e.g.nifedipine), folic acid or analogues. After polymerisation the polymeris freed from the template by a washing procedure. The template can alsocome from the group of dioxins (e.g. tetrachlorodioxodibenzene, TCDB),polyaromatic hydrocarbons (PAHs), aldehydes, ketones, halogenatedhydrocarbons and phosphonates (e.g. nerve agents like sarin, soman,tabun, VX). This procedure results in a polymer showing unprecedentedlyhigh selectivity and strong binding of the template or structurallyrelated compounds.

Example 26

[0127] The polymer prepared according to Example 25 can be used forseparation of enantiomers, diastereomers of the template or forseparation of the template or template analogues from structurallyrelated compounds present in a complex mixture. This can be done bychromatography, capillary electrophoresis, capillaryelectrochromatography, batch modes or membrane modes. The polymer canfurther be used for catalysing chemical reactions such as esterolysis,amidolysis, ester synthesis or amide synthesis.

LITERATURE

[0128] 1. R. A. Bartsch, M. Maeda, Eds., Molecular and Ionic Recognitionwith Imprinted Polymers (Oxford University Press, Washington, 1998).

[0129] 2. L. I. Andersson, R. Müller, G. Vlatakis, K. Mosbach, Mimics ofthe binding sites of opioid receptors obtained by, Proc. Natl. Acad.Sci. U.S.A. 92, 4788-92 (1995).

[0130] 3. B. Sellergren, Polymer- and template-related factorsinfluencing the efficiency in molecularly imprinted solid-phaseextractions, Trends Anal. Chem. 18, 164-174 (1999).

[0131] 4. B. Sellergren, M. Lepistoe, K. Mosbach, Highlyenantioselective and substrate-selective polymers obtained byinteractions. NMR and chromatographic studies on the nature ofrecognition, J. Am. Chem. Soc. 110, 5853-60 (1988).

[0132] 5. B. Sellergren, in A practical approach to chiral separation byliquid chromatography G. Subramanian, Ed. (VCH, Weinheim, 1994) pp.69-93.

[0133] 6. K. J. Shea, D. A. Spivak, B. Sellergren, Polymeric complementsfor adenine prepared by molecular imprinting, J. Am. Chem. Soc. 115,3368-3369 (1993).

[0134] 7. B. Sellergren, Direct drug determination by selective sampleenrichment on an imprinted polymer, Anal. Chem. 66, 1578 (1994).

[0135] 8. E. Fan, S. A. Van Arman, S. Kincaid, A. D. Hamilton, Molecularrecognition: Hydrogen bonding receptors that function in highlycompetitive solvents, J. Am. Chem. Soc. 115, 369-370 (1993).

[0136] 9. Lieser, Kemmner, Chem. Ber. 84, 4-12 (1951).

[0137] 10. Iwakura et.al. Jpn., 186-191 (1968), Bull. Chem. Soc. 41,186-191 (1968).

[0138] 11. e. a. Seiller, Tetrahedron 51, 10901-10912 (1995).

[0139] 12. Oikawa, J. Polym. Sci.: Part A: Polym. Chem. 31, 457-465(1993).

[0140] 13. D. Spivak, K. J. Shea, Molecular Imprinting of CarboxylicAcids Employing Novel Functional Macroporous Polymers, J. Org. Chem. 64,4627-4634 (1999).

[0141] 14. M. J. Kamlet, R. M. Doherty, J. -L. M. Abboud, M. H. Abraham,R. W. Taft, Solubility. A new look., Chemtech 566-576 (1986).

[0142] 15. M. C. Etter, T. W. Panunto, 1,3-Bis(m-nitrophenyl)urea: Anexceptionally good complexing agent for proton acceptors., J. Am. Chem.Soc. 110, 5896-5897 (1988).

[0143] 16. G. Wulff, R. Schönfelt, Polymerizablöamidines—Adhesionmediators and binding sites for molecular imprinting, Adv. Mater. 10,957-959 (1998).

[0144] 17. C. Dauwe, J. Buddrus, GIT-Fachzeitschrift für dasLaboratorium 38, 517 (1994).

[0145] 18. Fuks, Tetrahedron 29, 2147-2151 (1973).

[0146] 19. E. J. Roskamp, S. F. Pedersen, J. Am. Chem. Soc. 1957, 109,3152-3154.

1. A polymerisable functional monomer, wherein said monomer is a stronghydrogen bond donating monomer that is unable to function as a hydrogenbond acceptor, preferably a N,N′-disubstituted phenyl urea monomer ofthe formula

wherein X, Y and Z are independently selected from the group consistingof H, NO₂, CF₃ and halide, with the proviso that at least one of X and Yare not H.
 2. A molecularly imprinted polymer obtainable bypolymerisation of a monomer according to claims 1 and a cross-linkingmonomer in the presence of a template molecule.
 3. A polymer accordingto claim 2, wherein the template molecule is selected from the groupconsisting of a nucleotide or oligonucleotide, a polyelectrolyte such asa polysulfonic or polycarboxylic or mixed polysulfonic/polycarboxylicacid, a carboxylic acid, a phosphonic acid, a phosphoric or phosphinicacid, a sulfuric, sulfonic, sulfinic acid, an alcohol, an imide, athiol, a ketone, an amide, a sulfonamide, a phosphonamide, a hydantoin,a barbiturate, an ether, a polyaromatic hydrocarbon, a phosphonate, analdehyde.
 4. Use of a polymer according to claim 2 or 3, for separationof enantiomers, diastereomers of the template molecule.
 5. Use of apolymer according to claim 2 or 3, for separation of the templatemolecule or template molecule analogues from structurally relatedcompounds.
 6. Use of a polymer according to claim 2 or 3, for catalysingchemical reactions such as esterolysis, amidolysis, ester synthesis oramide synthesis.